The zebrafish has become the model system of choice for a growing number of investigators interested in understanding mechanisms of vertebrate development, disease and evolution. While forward genetic screens for mutations induced by chemical mutagenesis have uncovered the functions of many zebrafish genes, so far no reverse genetic technology has been developed that is both inexpensive and high-throughput. This application proposes to develop a method for identifying N-ethyI-N-nitrosourea (ENU)-induced mutations in genes of interest in zebrafish. The approach, termed TILLING, uses the CELl enzyme, which cuts DNA at the site of single basepair mismatches, to detect ENU-induced mutations in PCR-amplified genomic DNA fragments. Preliminary data shows that this approach can effectively identify ENU-induced mutations in zebrafish genomic DNA at a rate of 2.03 x 10" mutations per base pair, or one mutation per 493 kb. To determine the feasibility of this approach in zebrafish on a larger scale, a library will be constructed consisting of 10,000 ENU mutagenized fish, preserved as frozen sperm and genomic DNA, which can in principle be screened for mutations in any gene of interest. This library will be used to generate an allelic series including loss-of-function and reduction-of function mutations in 50 zebrafish genes. Targets include genes involved in embryonic patterning, with particular emphasis on genes that are hypothesized to control the patterning and segmentation of the developing hindbrain. In order to demonstrate the general applicability of this approach to the broader interests of the zebrafish community, mutations will also be identified in zebrafish genes that are inaccessible to three-generation forward genetic screens or antisense approaches currently available in the zebrafish. These include maternal effect genes, genes that are function in the adult nervous system, and putative tumor suppressor genes. The long-term objectives of the project are to optimize the efficiency of mutation detection by TILLING in zebrafish, to use mutations generated by TILLING to understand the genetic basis of zebrafish neural patterning, and above all to determine the feasibility of this approach as a general method for reverse genetics in zebrafish.